Development of an LC⁻Tandem Mass Spectrometry Method for the Quantitative Analysis of Hercynine in Human Whole Blood

• Published in: Molecules (Basel, Switzerland) Vol. 23; no. 12; p. 3326
• Main Authors: Sotgia, Salvatore, Murphy, Rhys B, Zinellu, Angelo, Elliot, David, Paliogiannis, Panagiotis, Pinna, Gerard Aimè, Carru, Ciriaco, Mangoni, Arduino A
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 14.12.2018; MDPI
• Subjects: Acetonitrile; Acids; aminothione; antioxidant; Biological activity; Biosynthesis; Blood levels; Calibration; Cold water; Crosstalk; Deuteration; dismutatiion; Erythrocytes; Formic acid; LC-tandem mass spectrometry; Lysis; Mass spectrometry; Mass spectroscopy; Metabolites; Oxidative stress; Physiology; Quantitative analysis; Retention; Scientific imaging; Water purification; zwitterion; dismutatiion; antioxidant; zwitterion; LC-tandem mass spectrometry; aminothione
• ISSN: 1420-3049; 1420-3049
• DOI: 10.3390/molecules23123326

Given that the peculiar redox behavior of ergothioneine involves a rapid regeneration process, the measurement of its precursor and redox metabolite hercynine could be particularly useful in assessing its role in oxidative stress or other biological processes. Thus, a LC-MS/MS method for the determination of hercynine concentrations in whole blood was developed. After lysis of red blood cells by cold water, samples were filtered on micro concentrators at a controlled temperature of 4 °C. The clear filtered fluid was then treated with diethylpyrocarbonate to derivatize hercynine for the analysis by LC-MS/MS. The derivatized analyte was isocratically separated as a carbethoxy derivative on a C18 column with a mobile phase of an aqueous 0.1% / formic acid and acetonitrile (95:5). Effluents were monitored by MRM transitions at / 270.28→95 and 273.21→95 for hercynine and its deuterated counterpart, respectively. No cross-talk between MRM transitions was observed and a good linearity was found within a range of 35⁻1120 nmol/L. The LOD and LOQ were, respectively, 10.30 and 31.21 nmol/L with an intraday and intermediate precision below 7%. The average hercynine concentration in whole blood from 30 healthy male volunteers (aged 77 ± 12 years) was 178.5 ± 118.1 nmol/L. Overall, the method is easy to perform, allowing a rapid and accurate assessment of whole blood concentrations of hercynine.